There is now compelling evidence that regulatory T cells (Tregs) suppress immune activation and are perturbed during HIV infection. Exploiting the mechanisms of Treg function is relevant for controlling chronic immune activation during HIV infection or conversely in boosting HIV-specific immune responses. However, several key questions and gaps remain in our understanding of Treg cell biology, especially in the context of HIV pathogenesis. The analysis of Tregs in HIV patients is complicated due to lack of specific markers. It is also still not clear whether Treg cell loss contributes to inflammation during HIV infection or whether they are detrimental by suppressing HIV-specific immune responses. During the previous grant period we made major advances in understanding Treg cell differentiation and established methods to expand them in vitro. We discovered a cell surface molecule, called GARP, highly specifically expressed on bona fide activated Tregs. We also found that receptors for proinflammatory cytokines IL-1 and TNF and their respective decoys are preferentially expressed on human Treg cells. Additionally, we developed a novel technology to expand and genetically engineer Tregs with HIV-specific T cell receptors (TCRs) to determine the role of TCR signals in their suppressive function. In this proposal we aim to use these innovative tools and technical advances to: (1) Determine association of Tregs with immune activation status of HIV-infected individuals either with or without treatment in a prospective study; (2) Determine the function of Tregs in sensing and suppressing inflammation and whether this function is compromised during HIV infection; (3) Determine the specificity and suppressive role of Tregs in manipulating HIV-specific immune responses; and (4) Develop an experimental framework to decode the role of peptide affinity and concentrations in regulating Treg responses through TCR signaling. Together, these new approaches will have wide range of implications for using bona fide Tregs as biomarkers during HIV infection, defining their role in immune activation and in suppressing HIV-specific immune responses during HIV infection. This knowledge can also be exploited to design more potent HIV vaccines.